JP2002171977A - New human sh2 protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new SH2 protein involved in a signal transduction through a tyrosine kinase, a gene encoding the protein and to provide a method for producing the protein and the gene and their uses. SOLUTION: Isolation of a DNA encoding a new protein HSH2 having an SH2 domain from a human placental tissue cDNA library is succeeded. It is suggested that the HSH2 is involved in a signal transduction system through tyrosine kinase, especially differentiation/proliferation and activation of blood system cells. The HSH2 is expected to be useful for developing an anticancer agent and medicines for hematopoietic /blood diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel SH2 protein, a gene encoding the protein, and production and use thereof.

[0002]

2. Description of the Related Art Cells respond appropriately to changes in the external environment and stimuli, such as changes in gene expression and intracellular skeleton, through various intracellular signal transduction pathways. Signal transduction pathways via tyrosine kinases play an important role in that. It has been found that signal transmission by this tyrosine kinase is transmitted by a series of protein interactions.

[0003] Among them, adapter proteins include various motifs such as SH2 (Src homology 2) domain, SH3
A group of proteins that play an important role in signal transduction via tyrosine kinases, which form protein complexes via domains, PH domains, proline-rich regions, tyrosine phosphorylation sites, etc. For example, the adapter protein Grb2
In its SH2 domain, it binds to phosphorylated tyrosine of various activated receptor tyrosine kinases,
Domains bind with SOS. As a result, SOS translocates from the cytoplasm to the cell membrane and activates Ras localized in the membrane. Activated Ras activates the MAP kinase pathway.

[0004] From these characteristics, the adapter protein is
It is an important target molecule for the development of drugs that control signaling through tyrosine kinase.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a novel SH2 protein involved in signal transduction via tyrosine kinases,
And a gene encoding the protein, and their production and use.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies as follows for the purpose of cloning a novel human gene. First, the oligo cap method (Maruyama K. a
nd Sugano S. Gene 138: 171-174, 1994; Suzuki Y. et
al. Gene 200: 149-156, 1997), a clone constituting a human cDNA library having a high full-length ratio was isolated. Next, the nucleotide sequence of the cDNA clone having a high total length obtained by this method was determined from both the 5 ′ side and the 3 ′ side, and ATGpr
(Salamov AA et al. Bioinformatics 14: 384-390,
1998; http://www.hri.co.jp/atgpr/) etc.
A human full-length cDNA that was expected to be a cDNA clone was selected. The full-length cDNA sequence of the selected clone was analyzed, and the amino acid sequence encoded by the nucleotide sequence was estimated. Then
Based on the deduced amino acid sequence, BLAST (Altschul SF
et al. J. Mol. Biol. 215: 403-410, 1990; Gish W.an
d States DJ Nature Genet. 3: 266-272, 1993; htt
According to p: //www.ncbi.nlm.nih.gov/BLAST/), GenBank (h
ttp: //www.ncbi.nlm.nih.gov/Web/Genbank/index.html)
And SwissProt (http://www.ebi.ac.uk/ebi_docs/swisspro
t_db / swisshome.html) to perform homology analysis,
A cDNA clone encoding the novel protein was selected. Using the nucleotide sequence of the human full-length cDNA clone thus obtained, PSORT (Nakai K and Kanehisa M. Genomics 14: 89
7-911, 1992), Pfam (http://www.sanger.ac.uk/Softwa
re / Pfam / index.shtml) and the like, and the signal sequence and functional domain were searched.

[0007] As a result, a clone C-PLACE1003723 having an SH2 domain and a proline-rich region was successfully obtained. The clone was presumed to be a gene encoding a novel SH2 protein.
I named it. From the results of the nucleotide sequence analysis, the HSH2 gene
It was 16 bp long and encoded a protein consisting of 352 amino acid residues.

[0008] As a result of Northern blot analysis, HSH2
Is expressed in leukocytes and spleen of peripheral blood, and was presumed to function in blood cells.

In addition, HSH2 by various tyrosine kinases
As a result of detecting protein phosphorylation, HSH2 was found to be c-AB
L, BCR-ABL, v-Src, JAK1, JAK2, and JAK3 were found to be phosphorylated. From this, HSH2
Was suggested to be involved in the signal transduction system via the tyrosine kinase as a substrate of the tyrosine kinase.

[0010] K562 established from patients with chronic myeloid leukemia
In cells, a fusion protein of BCR and ABL (BCR-ABL) is generated, and enhanced tyrosine kinase activity is thought to be the cause of canceration. Therefore, the present inventors examined the binding between tyrosine phosphorylated protein in K562 cells and HSH2. As a result, HSH2 was found to interact with phosphorylated proteins involved in signal transduction, such as BCR-ABL in K562 cells.

[0011] Then, next, the present inventors, the yeast Two Hy
A brid assay was used to determine whether HSH2 bound directly to BCR-ABL. As a result, HSH2 was found to directly bind to autophosphorylated BCR-ABL via its SH2 domain. Since the tyrosine kinase activity of BCR-ABL is essential for cell carcinogenesis, proteins phosphorylated by BCR-ABL or proteins interacting with it are considered to play an important role in leukemia formation. Therefore, the agent targeting HSH2 of the present invention is expected to act as an anticancer agent against cancer cells that cause leukemia.

[0012] Further, it was speculated that HSH2 functions in blood cells as described above. It is known that blood cells are differentiated / proliferated or activated by stimulation of various cytokines. At this time, a signal transduction system via tyrosine kinase is involved, and this signal transmission requires phosphorylation and interaction of various proteins. Then, the present inventors next examined the IL-3 receptor β chain and SHP2 phosphorylated by stimulation of the cytokine IL-3, and the HSH2 of the present invention.
Was examined for binding. As a result, it was found that the SH2 domain of HSH2 binds to the IL-3 receptor β chain whose tyrosine has been phosphorylated by IL-3 treatment and SHP2. As described above, considering that HSH2 is phosphorylated by various tyrosine kinases such as the JAK family activated by cytokines, it is considered that HSH2 is involved in the differentiation / proliferation and activation of blood cells. Can be Therefore, development of a drug for hematopoietic and hematologic diseases targeting HSH2 of the present invention is expected.

[0013] The present invention has been completed based on the above findings, the information transmission system via a tyrosine kinase,
In particular, a novel human SH2 protein "HSH
2 ", and a gene encoding the protein, and their production and use.

More specifically, (1) a DNA described in any one of the following (a) to (d), and (a) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. (B) DNA containing the coding region of the nucleotide sequence described in SEQ ID NO: 1. (C) a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and the amino acid sequence of SEQ ID NO: 2; DNA that encodes an equivalent protein. (D) a DNA which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2. (2) SEQ ID NO: 2
DNA encoding a partial peptide of a protein consisting of the amino acid sequence according to (3), (3) D according to (1) or (2),
A protein or peptide encoded by NA, (4)
A vector into which the DNA according to (1) or (2) has been inserted, (5) a transformed cell carrying the DNA according to (1) or (2) or the vector according to (4),
(6) Production of the protein or peptide according to (3), which comprises a step of culturing the transformed cell according to (5) and recovering the expressed protein or peptide from the transformed cell or a culture supernatant thereof. (7) an antibody that binds to the protein of (3), (8) a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof, (9) )
(3) A method for screening a compound that binds to a protein according to (3), wherein (a) a step of bringing a test sample into contact with the protein or a partial peptide thereof, and (b) a step of contacting the test sample with the protein or a partial peptide thereof. (C) selecting a compound having an activity of binding to the protein or a partial peptide thereof.
(10) A method for screening a compound having an activity of regulating the phosphorylation of the protein according to (3) by tyrosine kinase, comprising: (a) contacting the protein or a partial peptide thereof, a tyrosine kinase, and a test sample (B) detecting the phosphorylation of the protein or its partial peptide, (c) comparing the degree of phosphorylation detected in step (b) with the degree of phosphorylation detected in the absence of the test sample And selecting a compound to decrease or increase, (11) the tyrosine kinase is selected from the group consisting of c-ABL, BCR-ABL, v-Src, JAK1, JAK2, and JAK3, (10) A method for screening a compound having an activity of regulating the binding between the protein according to (3) and a tyrosine phosphorylated protein according to (12), A) contacting the protein or a partial peptide thereof, a tyrosine-phosphorylated protein, and a test sample; (b) detecting the binding of the protein or a partial peptide thereof to a tyrosine-phosphorylated protein; A) selecting a compound that decreases or increases the binding activity detected in the absence of the test sample, as compared to the case where the binding activity is detected in the absence of the test sample. (3) The method according to (12), which is selected from the group consisting of 3 receptor β chains, SHP2, BCR-ABL, CBL, and SHC.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a novel protein "HSH2"
I will provide a. The amino acid sequence of the human "HSH2" protein contained in the protein of the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of the cDNA encoding the protein is shown in SEQ ID NO: 1. "HSH2"
Due to its structural characteristics, proteins are thought to play a role related to the tyrosine kinase-mediated signal transduction system, particularly the differentiation / proliferation and activation of blood cells, as an adapter protein in vivo. As a target for drug development of diseases related to these life phenomena including cancer,
Alternatively, it is expected to be applied as a drug itself.

The protein of the present invention can be prepared as a recombinant protein or as a natural protein. The recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention is inserted into an appropriate host cell as described below, and purifying the protein expressed in the transformant. . On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular B
iology edit. Ausubel et al. (1987) Publish. John W
iley & Sons Section 16.1-16.19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. In addition, in vitro translation (for example, “On the fidelity of mRNA”
translation in the nuclease-treated rabbit reticul
lysate system.Dasso, MC, Jackson, RJ (1989)
Nucleic Acids Res. 17: 3129-3144 ”) and the like, and the protein of the present invention can be prepared.

The present invention includes a protein functionally equivalent to the human-derived "HSH2" protein identified in this Example. Such proteins include, for example, SEQ ID NO: 2
And mutants, homologues, and variants of the “HSH2” protein described in (1). In the present invention, "functionally equivalent" means that the target protein is c-ABL, BCR-ABL, v-Src, JAK
1.Activity phosphorylated as a substrate for tyrosine kinases such as JAK2 and JAK3, or IL-3 receptor β chain, SHP2, BC
It refers to having the activity of binding to tyrosine phosphorylated proteins such as R-ABL, CBL, and SHC. Whether or not the target protein is phosphorylated by tyrosine kinase can be determined, for example, by introducing a plasmid expressing the target protein and a plasmid expressing tyrosine kinase into cells, as shown in Example 8, and The determination can be made by preparing a cell extract and performing Western blot analysis on the cell extract using an anti-phosphorylated tyrosine antibody.
In addition, the binding between the target protein and the tyrosine phosphorylated protein is determined, for example, by using Two Hybrid as described in Example 11.
According to the assay, and as shown in Example 9, a vector expressing the target protein was introduced into cells such as BaF3 cells, and the cells were stimulated with IL-3, and the target protein was recovered from the cell lysate. The determination can be made by detecting the tyrosine phosphorylated protein bound to the target protein using an antibody against the phosphorylated tyrosine.

Those proteins functionally equivalent to the proteins identified in this example can be prepared by those skilled in the art, for example,
Methods for introducing a mutation into an amino acid sequence in a protein (for example, site-directed mutagenesis (Current Protocols in Mole
cular Biology edit. Ausubelet al. (1987) Publish.
It can be prepared using John Wiley & Sons Section 8.1-8.5)). In addition, such proteins may be caused by amino acid mutations in nature. According to the present invention, one or more amino acids may be substituted, deleted, inserted and / or added in the amino acid sequence (SEQ ID NO: 2) as long as the protein has a function equivalent to the protein identified in this example. Different proteins are included depending on the conditions.

The number and location of amino acid mutations in a protein are not limited as long as the function is maintained. The number of mutations is typically within 30 amino acids, preferably 10
It is within amino acids, more preferably within 5 amino acids (eg, within 3 amino acids). The amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution from the viewpoint of maintaining the function of the protein. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as non-polar amino acids, and thus are considered to have similar properties. Gly, S
er, Thr, Cys, Tyr, Asn, and Gln. Examples of acidic amino acids include Asp and Glu,
Basic amino acids include Lys, Arg, and His.

Further, a protein functionally equivalent to the protein identified in the present example is obtained by a hybridization technique (Current Protocols in Molecular Biology) well known to those skilled in the art.
ology edit. Ausubel et al. (1987) Publish. John Wi
ley & Sons Section 6.3-6.4) or Gene amplification technology (PCR) (Current protocols in Molecular Biology)
edit. Ausubel et al. (1987) Publish. John Wiley &
It can also be isolated using Sons Section 6.1-6.4). That is, those skilled in the art can use a DNA encoding the protein identified in the present example (SEQ ID NO: 1) or a part thereof as a probe, or an oligonucleotide that specifically hybridizes with the DNA as a primer, DNA that hybridizes with the DNA can be isolated. Further, based on the isolated DNA, a protein encoded by the DNA can be prepared. The present invention includes proteins encoded by DNAs that hybridize to DNAs encoding these proteins, as long as they have the same function as the proteins identified in the present examples. Organisms from which functionally equivalent proteins are isolated include, but are not limited to, vertebrates such as humans, mice, rats, rabbits, pigs, and cows.

The stringent conditions for hybridization for isolating a DNA encoding a functionally equivalent protein are usually about 1 × SSC, 0.1% SDS, and 37 ° C. xSSC, 0.1% SDS, 4
2 ° C ”, and the more severe condition is“ 0.1xSS
C, 0.1% SDS, 65 ° C. ”, and isolation of DNA having higher homology to the probe sequence can be expected as the hybridization conditions become more stringent. However, the above SSC, SDS
The combination of the temperature and temperature conditions is merely an example, and those skilled in the art may appropriately combine the above or other factors (eg, probe concentration, probe length, hybridization reaction time, etc.) that determine the stringency of hybridization. Thereby, the same stringency as described above can be realized.

Proteins isolated using such a hybridization technique or a gene amplification technique are as follows:
Compared with the protein of the present invention described in SEQ ID NO: 2, it usually has higher homology in its amino acid sequence. High homology means at least 50% or more, more preferably
It refers to 70% or more, more preferably 90% or more (eg, 95% or more) sequence identity. Sequence homology was determined by BLAST X
Can be determined using a homology search by As a sequence to be compared, a region having a length of 100 or more residues in the amino acid sequence of 352 residues (human) of the HSH2 protein can be used.

The present invention also provides a partial peptide of the protein of the present invention. The partial peptide of the protein of the present invention,
For example, it can be used for screening a compound that binds to the protein of the present invention described below, and for preparing an antibody that binds to the protein of the present invention. The partial peptide of the present invention has at least 7 amino acids, preferably 9 amino acids or more,
More preferably, it comprises an amino acid sequence of at least 12 amino acids, more preferably at least 15 amino acids. When used for screening a compound that binds to the protein of the present invention or a compound having an activity of regulating the binding between the protein of the present invention and a tyrosine kinase, a peptide containing the SH2 domain of HSH2 is used as a partial peptide. Can be used. Preferably, it is a partial peptide containing the amino acid sequence from the 34th tryptophan to the 124th proline in the amino acid sequence of the HSH2 protein (SEQ ID NO: 2). When used for screening for a compound that regulates the phosphorylation of the protein of the present invention by tyrosine kinase described below, the partial peptide may be HS.
A partial peptide containing an H2 phosphorylation site can be used.

Similarly, when used for screening for a compound that binds to the protein of the present invention, a peptide containing a proline-rich region of HSH2 can be used as a partial peptide. Preferably, in the amino acid sequence of the HSH2 protein (SEQ ID NO: 2), an amino acid sequence from proline 8 to proline 13; an amino acid sequence from proline 191 to proline 195; The amino acid sequence up to the th proline,
Alternatively, it is a partial peptide containing any region of the amino acid sequence from proline 344 to proline 349.

The partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.

The present invention also provides a DNA encoding the protein or peptide of the present invention. The DNA of the present invention is not particularly limited in its form as long as it can encode the protein or peptide of the present invention, and includes cDNA, genomic DNA, chemically synthesized DNA, and the like. In addition, DNAs having an arbitrary base sequence based on the degeneracy of the genetic code are included as long as they can encode these molecules. As described above, the DNA of the present invention can be prepared by a hybridization method using the DNA sequence of SEQ ID NO: 1 or a part thereof as a probe,
It can be isolated by a conventional method such as a gene amplification method (PCR) using a primer designed based on the information of the DNA sequence.

[0027] The present invention also provides a vector into which the DNA of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it can stably maintain the inserted DNA.For example, if Escherichia coli is used as a host, a pBluescript vector (Strata
gene). When a vector is used for producing the protein of the present invention, an expression vector is particularly useful. As an expression vector, for example, pBEST vector (Promega) for in vitro expression
Is expressed in Escherichia coli, the pET vector (Invitro
gen) is pME18S-FL if expressed in cultured cells
3 If the vector (GenBank Accession No. AB009864) is expressed in an individual organism, the pME18S vector (Mol Cell
Biol. 8: 466-472 (1988)). Insertion of the DNA encoding the protein of the present invention into a vector is performed by a conventional method, for example, a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. A).
usubel et al. (1987) Publish. John Wiley & Sons. S
Sections 11.4 to 11.11).

The present invention also provides a transformant carrying a vector into which a DNA encoding the protein of the present invention has been inserted. The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells may be used depending on the purpose. Host cells can be used, for example, for production of the proteins of the invention. Production systems for protein production include in vitro and in vivo production systems. in vit
Examples of ro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. Further, the host cells of the present invention include:
It also includes cells of interest for use in functional analysis of "HSH2" protein and screening for its function inhibitors and function promoters utilizing "HSH2" protein. For example, the introduction of a vector into a host cell is performed by, for example, a calcium phosphate precipitation method or an electric pulse perforation method (Current protocols in Molecular Biology edit.
Ausubel et al. (1987) Publish. John Wiley & Son
s. Section 9.1-9.9), Lipofectamine method (GIBCO-BR)
L), a microinjection method, or the like. The "HSH2" protein can be prepared from the transformant using a protein separation / purification method known to those skilled in the art.

The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof.
Here, the “complementary strand” refers to one strand of a double-stranded DNA composed of A: T, G: C base pairs with respect to the other strand. Further, the term "complementary" is not limited to a case where the sequence is completely complementary in at least 15 contiguous nucleotide regions.
70%, preferably at least 80%, more preferably 90%
More preferably, it should have at least 95% or more homology on the base sequence. The algorithm described in the present specification may be used as an algorithm for determining homology.

Such a polynucleotide can be used as a probe for detecting and isolating a DNA encoding the protein of the present invention, and as a primer for amplifying the DNA of the present invention. When used as a primer, usually 15 bp to 100 bp, preferably 15 bp
It has a chain length of p-35 bp. When used as a probe, a DNA having at least a part or all of the sequence of the DNA of the present invention and having a chain length of at least 15 bp is used. When used as a primer, the 3′-side region needs to be complementary, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5′-side.

The polynucleotide of the present invention can be used for testing and diagnosing abnormalities of the protein of the present invention. For example, abnormal expression can be examined by Northern hybridization using the polynucleotide of the present invention as a probe or primer, or by RT-PCR.
Furthermore, by genomic DNA-PCR or RT-PCR using the polynucleotide of the present invention as a primer, the DNA encoding the protein of the present invention and its expression control region are amplified, followed by RFLP analysis, SSCP, sequencing, etc. By doing so, abnormalities in the sequence can be inspected and diagnosed.

Further, the polynucleotide of the present invention includes an antisense for suppressing the expression of the “HSH2” protein of the present invention. The antisense is at least 15 bp or more, preferably 10 bp or more, to cause an antisense effect.
It has a chain length of 0 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less. Such antisense is performed, for example, based on the phosphorothioate method (Stein, 1988 Physicochemi) based on the sequence information of DNA described in SEQ ID NO: 1.
cal properties of phosphorothioate oligodeoxynucle
otides. Nucleic Acids Res 16, 3209-21 (1988)).

The DNA of the present invention and its antisense can be applied to, for example, gene therapy. As a disease targeted for gene therapy, for example, cancer and various inflammatory diseases are considered to be suitable. When these molecules are used for gene therapy, for example, a retroviral vector, an adenovirus vector, a virus vector such as an adeno-associated virus vector, a non-viral vector such as a liposome, or the like, using an ex vivo method or an in vivo method, etc. May be administered to the patient.

The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. Also, all classes of antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

In the case of a polyclonal antibody, the antibody of the present invention can be obtained by synthesizing an oligopeptide corresponding to the amino acid sequence according to a conventional method and immunizing a rabbit (Current protocols in Molecular Biology e).
dit. Ausubel et al. (1987) Publish. John Wiley & So
ns. Section 11.12-11.13) On the other hand, in the case of monoclonal antibodies, mice were immunized with a protein expressed and purified in E. coli according to a conventional method, and hybridoma cells obtained by fusing spleen cells and myeloma cells were used. (Current protocols in Molecular Biology edit.
Ausubel et al. (1987) Publish. John Wiley & Sons.
Section 11.4-11.11).

Antibodies that bind to the protein of the present invention may be used, for example, for examination and diagnosis of abnormal expression or structural abnormality of the protein in addition to purification of the protein of the present invention. Specifically, for example, a protein is extracted from tissue, blood, cells, or the like, and the presence or absence of abnormal expression or structure is examined and diagnosed through detection of the protein of the present invention by a method such as Western blotting, immunoprecipitation, or ELISA. be able to.

Antibodies that bind to the protein of the present invention may be used for purposes such as treatment of diseases related to the protein of the present invention. When an antibody is used for the purpose of treating a patient, a human antibody or a humanized antibody is preferred because of its low immunogenicity. Human antibodies are obtained by replacing the immune system with that of a human (eg, “Functional transplant of megab
ase human immunoglobulin loci recapitulates human
antibody response inmice, Mendez, MJ et al. (199
7) Nat. Genet. 15: 146-156 ”). Further, a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203,
99-121 (1991)).

[0038] The present invention also provides a method for screening for a compound that binds to the protein of the present invention. This screening method comprises (a) a step of bringing a test sample into contact with the protein of the present invention or a partial peptide thereof, (b) a step of detecting a binding activity between the protein or a partial peptide thereof and the test sample, and (c) And b) selecting a compound having an activity of binding to the protein or a partial peptide thereof.

The test sample is not limited to these, but includes, for example, a cell extract, an expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, and the like. The protein of the present invention or its partial peptide used for screening may be labeled as necessary, and may be fused with another protein or peptide for the purpose of facilitating purification of the protein of the present invention. May be. In this screening method,
When a two-hybrid system is used, the protein of the present invention can be used in the form of a fusion protein with a transcription activation region or a DNA binding region of a transcription factor.

As a specific method, there are many known methods such as a method of bringing a test sample into contact with an affinity column of the protein of the present invention to purify the protein, a West-Western blotting method, a method by high-throughput screening in a combinatorial chemistry technique, and the like. Can be used. BIACORE (Pharmacia)
Screening can also be performed by evaluating the binding between the protein of the present invention and a test compound using a measuring device such as the above.

The compound that binds to the protein of the present invention isolated by this screening is a candidate for a compound (agonist, antagonist) that promotes or inhibits the activity of the protein of the present invention. In addition, in a living body, the protein of the present invention is a candidate for a compound that inhibits the interaction between the protein and a molecule that interacts with the protein.

The present invention also provides a method for screening for a compound having an activity of regulating the phosphorylation of the protein of the present invention by tyrosine kinase. This screening method comprises the steps of (a) contacting the protein of the present invention or its partial peptide, tyrosine kinase, and a test sample, (b) detecting phosphorylation of the protein or its partial peptide, (c) step Selecting a compound that reduces or increases the degree of phosphorylation in (b) as compared to the case where the degree of phosphorylation is detected in the absence of the test sample.

As the test sample, for example, a cell extract,
An expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, or the like can be used, and a compound that binds to the protein of the present invention obtained by the above screening can also be used. The tyrosine kinase is not particularly limited as long as it can phosphorylate the protein of the present invention, for example, c-ABL, BCR-ABL, v-
Src, TAK1, TAK2, TAK3 and the like can be used. In screening, instead of the protein of the present invention,
A partial peptide of the protein of the present invention containing a phosphorylation site of tyrosine kinase may be used.

This screening can be performed, for example, as follows. First, cells into which a vector expressing the protein of the present invention as a fusion protein with a tag and a vector expressing a tyrosine kinase are prepared. The tag is not particularly limited as long as it facilitates purification of the protein of the present invention, and for example, GST or the like can be used. Next, a test sample is brought into contact with the cells, and the protein of the present invention is purified from a lysate of the cells using a tag as an indicator. Next, phosphorylation of the protein of the present invention is detected by performing Western blot analysis on the purified protein of the present invention using an anti-phosphorylated tyrosine antibody. Compare the results of the detection with those of a control (in which a similar experiment is performed without adding a test sample to detect the phosphorylation of the protein of the present invention) to reduce the degree of phosphorylation of the protein of the present invention or Select compounds to increase.

As another method, for example,
And a tagged protein of the present invention,
Purified tyrosine kinases (c-ABL, BCR-ABL, etc.)
To ³²-P radioisotope and test sample
Material is brought into contact with the protein of the present invention.³²-P measured
To determine the activity of tyrosine kinases
Is mentioned.

As a result, the compound that reduces the degree of phosphorylation of the protein of the present invention was determined to have the activity of inhibiting the phosphorylation of the protein of the present invention by tyrosine kinase. Are determined to have an activity of promoting the phosphorylation of the protein of the present invention by tyrosine kinase.

The present invention also provides a method for screening for a compound having an activity of regulating the binding between the protein of the present invention and a tyrosine phosphorylated protein. This screening includes (a) a step of contacting the protein of the present invention or its partial peptide, a tyrosine-phosphorylated protein, and a test sample, and (b) detecting the binding between the protein or its partial peptide and a tyrosine-phosphorylated protein. And (c) selecting a compound that reduces or increases the degree of binding in step (b) as compared to that detected in the absence of the test sample.

As the test sample, for example, a cell extract,
An expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, or the like can be used, and a compound that binds to the protein of the present invention obtained by the above screening can also be used. The tyrosine phosphorylated protein is not particularly limited as long as it can bind to the protein of the present invention, for example, IL-3 receptor β chain,
Examples include, but are not limited to, SHP2, BCR-ABL, CBL, SHC, and the like. In the screening, a partial peptide of the protein of the present invention containing the phosphorylation site of the tyrosine phosphorylated protein may be used instead of the protein of the present invention. The partial peptide is preferably a partial peptide containing an SH2 domain.

This screening can be performed as follows. For example, when the tyrosine phosphorylated protein is a tyrosine kinase, it can be carried out by a method using a two hybrid system. In this method, first, a DNA encoding the protein of the present invention and a DNA encoding a tyrosine phosphorylated protein are inserted into two vectors constituting the system, respectively, and are introduced into cells such as yeast. Next, a test sample is brought into contact with the cells, and the reporter activity in the cells is detected (the reporter activity is an indicator of the degree of binding between the protein of the present invention and the tyrosine phosphorylated protein). The result of the detection is compared with that of a control (when a similar experiment is performed without adding a test sample to detect a reporter activity), and a compound that decreases or increases the reporter activity is selected.

In the case of a tyrosine phosphorylated protein other than tyrosine kinase, screening can be performed, for example, by the following method. in this way,
The purified protein of the present invention with a tag is mixed with a cell extract (lysate) of a cell (a cell stimulated with IL-3 or a cell into which BCR-ABL has been introduced) contacted with a test sample. The binding activity of the tyrosine phosphorylated protein in the extract to the protein of the present invention is examined. Alternatively, the purified protein of the present invention with a tag, the purified tyrosine phosphorylated protein, and a test sample are mixed, and the binding between the protein of the present invention and the tyrosine phosphorylated protein is examined. The result of the detection is compared with that of a control (in which a similar experiment is carried out without adding a test sample to detect the binding activity), and a compound that reduces or increases the binding activity is selected.

As a result, the compound that reduces the degree of binding between the protein of the present invention and the tyrosine phosphorylated protein was determined to have the activity of inhibiting the binding between the protein of the present invention and the tyrosine phosphorylated protein. A compound that increases the degree of binding between the protein of the present invention and a tyrosine phosphorylated protein is determined to have an activity of promoting the binding between the protein of the present invention and the tyrosine phosphorylated protein.

The protein or gene of the present invention or the compound isolated by the above screening may be used for controlling the differentiation, proliferation and activation of blood cells. For example, it is expected that these molecules can be used to treat cancer, hematopoietic and blood diseases, and the like.

When these molecules are used as pharmaceuticals, they can be used as pharmaceuticals themselves, but they can also be formulated and used by known pharmaceutical methods. For example, it is conceivable to formulate a suitable combination with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like. Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. If the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art.

[0054]

EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. [Example 1] Preparation of cDNA library by oligocap method From human placental tissue (PLACE1), literature (J. Sambrook, E.
F. Fritsch & T. Maniatis, Molecular Cloning Second
edition, Cold Spring harbor Laboratory Press, 198
9) mRNA was extracted by the method described. Furthermore, oligo d
Poly (A) + RNA was purified on T-cellulose.

From each poly (A) + RNA, oligocap method [M. Maruyama and S. Sugano, Gene, 138: 171-174]
(1994)]. Oligo-cap
linker (5'-agcaucgagu cggccuuguu ggccuacugg-
3 ′; SEQ ID NO: 3) and Oligo dT primer (5′-gcg
gctgaag acggcctatg tggccttttt tttttttttt tt-3 ';
References [SEQ ID NO: 4) [Suzuki and Sugano, Protein Nucleic Acid Enzyme, 41: 197-201 (1996), Y. Suzuki et al., Ge
ne, 200: 149-156 (1997)].
terial Alkaline Phosphatase) treatment, TAP (Tobacco Ac
id Phosphatase) treatment, RNA ligation, first strand cDN
A synthesis and RNA removal were performed. Next, a 5 ′ PCR primer (5′-agcatcgagt cggccttgtt g-3 ′; SEQ ID NO: 5) and a 3 ′ PCR primer (5′-gcggctgaag acggcc
PCR (polymerase ch) using tatg t-3 ′; SEQ ID NO: 6)
The DNA was converted to double-stranded cDNA by ain reaction and cut with SfiI. Next, pME18SFL3 (GenBank AB009864, Expressio
n vector) (PLACE1) and the direction of the cDNA was determined and cloned to prepare a cDNA library. For the plasmid DNA of the clone obtained from these, the 5 'end of the cDNA or
The base sequence at the 3 'end is converted to a DNA sequencing reagent (Dye Termina
tor Cycle Sequencing FS Ready Reaction Kit, dRhoda
mine Terminator Cycle Sequencing FS Ready Reaction
Kit or BigDye Terminator Cycle Sequencing FS Re
After performing a sequencing reaction using an ady Reaction Kit (manufactured by PE Biosystems) according to the manual, the DNA base sequence was analyzed using a DNA sequencer (ABI PRISM 377, manufactured by PE Biosystems).

The oligocap high-full-length cDNA library was constructed using the expression vector pME18SFL3 capable of expression in eukaryotic cells.
It was produced using. pME18SFL3 has an SRα promoter and an SV40 small t intron incorporated upstream of the cloning site, and an SV40 polyA addition signal sequence inserted downstream thereof. Since the cloning site of pME18SFL3 is an asymmetric DraIII site and a complementary SfiI site is added to the end of the cDNA fragment,
The cloned cDNA fragment is inserted unidirectionally downstream of the SRα promoter. Therefore, in a clone containing the full-length cDNA, the gene can be transiently expressed by directly introducing the obtained plasmid into COS cells. That is, it is very easy to experimentally analyze proteins as gene products or their biological activities.

[Example 2] Evaluation of full length of 5'-end of clone from cDNA library prepared by oligo cap method
A method using a program developed by AA Salamov, T. Nishikawa, MB Swindells of the Helix Research Institute (ATGpr, ATGpr2;
It was determined in Japanese Patent Application No. 11-248036). For all clones whose 5'-terminal sequence matches the known human mRNA in the public database, if the 5'-terminal is longer than the known mRNA sequence in the public database, or if the 5'-terminal is shorter but the translation initiation codon is Was determined to be "full length", and the case not including the translation initiation codon was determined to be "non-full length". The 5'-end full-length ratio of the cDNA clone [full-length clone number / (total length clone number + non full-length number clones) in each library began more be compared with human known mRNA.

Next, the clones were evaluated using ESTiMateFL. ESTiMateFL is EST 5'- in public database
Nishikawa of Helix Research Institute selects clones with high possibility of full-length cDNA by comparison with terminal sequence and 3'-terminal sequence.
This method was developed by Ota et al.

According to this method, when an EST extending longer than the 5'-end or 3'-end sequence of a certain cDNA clone exists, the clone is judged to be "possibly not full-length". It is systematized so that it can be processed in large quantities by a method. Even if the 5'-end is longer than the EST sequence in the public database, or even if the clone has a shorter 5'-end, the difference is within 50 bases for the sake of convenience. And In the case of the 5'-end sequence of a clone hit with a known mRNA, about 80% of the sequence estimated to be full-length by EST is also full-length by evaluation of the 5'-end sequence for the known mRNA. Approximately 80% of the 5'-terminal sequences evaluated as non-full length were evaluated for a known mRNA, but the 5'-terminal sequence was non-full length. In the evaluation of full-length likelihood by comparison with ESTs, if the number of ESTs to be compared is large, the prediction accuracy is improved, but if the number of target ESTs is small, there is a drawback that the reliability of the prediction result is reduced. This method is effective if it is used to exclude clones that are likely not to be full-length from oligocapped cDNA clones having a total length ratio of about 60% at the 5'-terminal sequence. Also, ESTiMate
FL is a particularly effective method for evaluating the full length of the 3'-terminal sequence of cDNA of human unknown mRNA having an appropriate number of EST entries in public databases.

Using both ATGpr and ESTiMateFL programs, 18,959 clones whose 5′-terminal sequence of a clone from a human cDNA library prepared by the oligocap method was identical to known human mRNA were evaluated. That is, for the 5'-terminal sequence of a clone corresponding to a known human mRNA, ATG
The maximum value of pr1 and the 5'-terminal sequence of each clone
Compared to the NA ORF, it was determined whether it was full length or non-full length. Next, the 5′-end sequence of each clone was analyzed by ESTiMateFL to determine whether it was full length or non-full length. Specifically, for a clone from a human cDNA library prepared by an oligocap method in which the 5'-terminal sequence matches a known human mRNA, ES
The EST sequence was compared with the 5'-terminal sequence of the clone by TiMateFL, and a clone other than the non-full length EST was selected.

Example 3 Characterization of Clone Sequence The 5′-terminal sequence data of the clone sequence was characterized by the following method. (1) By homology search using BLAST for GenBank, it is confirmed whether it is identical to the mRNA sequence of human or other organisms (including the licensed sequence) or the human EST sequence. (2) Check whether the 5 'end is longer than the human mRNA sequence or human EST sequence. (3) ATGpr, ATGpr2 program for predicting full length
ATGpr1, derived from all start codons in the 5 'terminal sequence,
Determine the ATGpr2 value. (4) Determine the number of identical human EST sequences by homology search using BLAST for GenBank.

The characterization of the 3 ′ terminal sequence data of the clone sequence was carried out for the above (1) and (4). A final clone was selected based on the data of the clone sequences subjected to these characterizations.

Example 4 Identical EST by homology search
Prediction of novelty from the number of sequences The number of EST sequences that became the same as the EST sequence for each of the 5 'terminal sequence and the 3' terminal sequence was determined by homology search using GenBank. The human EST sequence was determined to be identical when the length of the comparison sequence portion with the 5 ′ terminal sequence was 90% or more over 200 bases or more. The number of EST sequences was directly used for characterization and used as an index of novelty. A clone having a 5 'terminal sequence and a 3' terminal sequence which are not identical to not only the mRNA sequence but also the EST sequence is a gene encoding a novel protein. Similarly, a clone having the same 5 'terminal sequence or a 3' terminal sequence having a small number of EST sequences was also determined to be a gene encoding a novel protein.

Example 5 Search for Signal Sequence, Transmembrane Region and Functional Domain for Deduced Amino Acid Sequence The presence or absence of the amino terminal signal sequence and the transmembrane region for the amino acid sequence deduced from the full-length nucleotide sequence of the novel gene Was predicted, and a functional domain (motif) of the protein was searched. For the amino-terminal signal sequence, see PSORT [K. Nakai and M. Kanehisa, Genomics, 14: 89
7-911 (1992)], and SOSUI [T.Hiro
kawa et.al. Bioinformatics, 14: 378-379 (1998)]
(Mitsui Information Development Co., Ltd. sales) was used for the analysis.
For search of functional domains, please refer to Pfam (http: //www.sange
r.ac.uk/Software/Pfam/index.shtml) was used.

As a result, a novel protein having the SH2 domain (clone C-PLACE1003723) was found, and this novel protein was named HSH2. From the results of the nucleotide sequence analysis, the HSH2 gene was 1916 bp in length and encoded a protein having a length of 352 amino acid residues. As shown in FIG. 1, the HSH2 protein was found to have Src homology domain 2 at the N-terminus (underline in FIG. 1). It also had four proline-rich regions to which the SH3 domain could bind (circle in FIG. 1) and five tyrosine residues that were phosphorylated and could bind to other proteins (circle in FIG. 1). Circle).

Example 6 HS by Northern Blot
Expression analysis of H2 The expression of HSH2 was examined by Northern blot analysis. Human
Using a multi-tissue Northern blot (Clontech)
^{Use a 32} P-labeled human HSH2 cDNA probe at 65 ° C
Prehybridization and hybridization were performed in pressHyb hybridization solution (Clontech). The blot was washed twice in a solution containing 0.3 M NaCl, 0.03 M sodium citrate, and 0.1% sodium dodecyl sulfate (SDS) for 1 hour at room temperature. Then, 15 mM NaCl,
In a solution containing 1.5 mM sodium citrate, 0.1% SDS, 6
After washing at 5 ° C for 1 hour, analysis was performed by autoradiography.

As a result, it was strongly expressed in peripheral blood leukocytes (FIG. 2). It was also expressed in the spleen. When expression was examined by RT-PCR, it was strongly detected in the peripheral blood and spleen, and weakly detected in other organs. This suggests that HSH2 functions in blood cells.

Example 7 Construction of Plasmid The plasmid used in the following examples was prepared as follows. pcDNA3-mys-HSH2 was constructed by inserting a full-length HSH2 cDNA fragment into the EcoRI and XhoI sites of pcDNA-myc.
The full-length HSH2 cDNA fragment was prepared using the C-PLACE1003723 clone as a template, and using a 5′-GAGA GAA TTC ATG ACA GAG GCC GGG AAG CT
G-3 '(SEQ ID NO: 7), and 5'-GAGA CTC GAG CTA
PCR was performed using GCA GTA CCC AGG GGC AAA-3 ′ (SEQ ID NO: 8) as a primer, and digested with EcoRI and XhoI. pcDNA3 c-ABL, pcDNA3p185BCR-ABL, and pcDNA3 v-Src were constructed by subcloning cDNA into the EcoRI site of pcDNA3. pME18S JAK1, pSRa-bsr JAK
2, pSRa-bsr JAK3 was obtained from the Institute of Medical Science, The University of Tokyo. pGEX SH2 / HSH2 was constructed by inserting the HSH2 cDNA fragment encoding the SH2 domain into the EcoRI and XhoI sites of pGEX 4T-1. The HSH2 cDNA fragment was prepared using 5′-GAGA GAA TTC ATG ACA GAG GCC GGG AAG using C-PLACE1003723 as a template.
CTG-3 '(SEQ ID NO: 9), and 5'-GAGA CTC GAG CTA
CTC GTA ATC CAC GTT TGC GGG-3 '(SEQ ID NO: 10)
Perform PCR using as primers EcoRI and XhoI
And digested. pBTM185bcr-abl WT and pBTM18
5bcr-abl KD has wild-type p185bcr-ab in pBTM116, respectively.
and cDNA encoding p185bcr-abl deficient in kinase activity. pVP16 CrkL and pVP16 Gr
b2 was constructed by inserting CrkL cDNA and Grb2 cDNA into pVP16, respectively. The pGAD20 plasmid is the XhoI of pGAD10.
Oligonucleotides annealed to the EcoRI site
5'-T CGA CTG GAA TTC GGA TCC GTC GAC CTC GAG A-3 '
(SEQ ID NO: 11), and 5'-AA TTT CTC GAG GTC G
It was constructed by inserting AC GGA TCC GAA TTC CAG-3 '(SEQ ID NO: 12). pGAD-HSH2, pGAD-SH2 / HSH2, and pGAD
-ΔSH2 / HSH2 was constructed by inserting the corresponding cDNA into the EcoRI and XhoI sites of pGAD20. The fragment corresponding to ΔSH2 / HSH2 was a C-PLACE1003723 clone, as well as 5′-GAGA GAA
TTC CCCGCA AAC GTG GAT TAC GA-3 '(SEQ ID NO: 1
3), and 5'-GAGA CTC GAG CTA CTC GTA ATC CAC G
It was amplified by PCR using TT TGC GGG-3 ′ (SEQ ID NO: 14) as a primer.

Example 8 HS by Tyrosine Kinase
H2 phosphorylation Plasmid was introduced into COS7 cells, and Myc-tagged HSH2
And various tyrosine kinases. Plastic used
Sumid (pcDNA3-mys-HSH2, pcDNA3 c-ABL, pcDNA3 p185B
CR-ABL, pcDNA3 v-Src, pME JAK1, pME JAK2, pME JAK
The construction method of 3) is shown in Example 7. Cell culture, and
Transfection was performed as follows. COS7 fine
Vesicles with 10% fetal bovine serum (FBS), penicillin, and
Dulbecco's Modified Eagle with Streptomycin
The cells were cultured in a medium (DMEM). Cells are transfected
Of the 35mm multi-well plate (Falcon)
2.0 × 10 per well^FiveFor about 16 hours. Plasmid
2 μg of lipofectamine (G
IBCO BRL) Preincubate with 13 μl at room temperature for 45 minutes
I was vate. Cells were washed once with DMEM. Plain cue
The baited mixture is diluted with DMEM to a final volume of 1 ml and
Vesicles. The cells are then cultured at 37 ° C for 5 hours,
FBS was added to a concentration of 10%. Transfection 2
After 4 hours, cells are washed once with DMEM, and added with DMEM-10% FBS
Was incubated with DMEM-1% BSA for 24 hours. Troff
48 hours after injection, cells are washed twice with cold PBS and NP
40 lysis buffer (20mM Tris [pH 8.0], 1mM ethylenedi
Amine tetraacetate (EDTA), 150mM NaCl, 1% NP-
40, 10% glycerol) dissolved in 150 μl,
Incubated for 20 minutes. Cell lysate 1.5 ml tube
And centrifuged. Then analyze the supernatant by SDS-PAGE
did. K562 cells were obtained from FBS, penicillin, and strep.
The cells were cultured in RPMI1640 medium supplemented with tomycin. BaF3 fine
Vesicle is recombinant mouse interleukin 3 (IL3)
And cultured in RPMI1640 medium containing

Subsequently, a cell extract was prepared and subjected to Western blot analysis using an anti-phosphorylated tyrosine antibody. Western blot analysis was performed as follows.

The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane (Biorad). Binding residue sites were blocked by incubating overnight at 4 ° C. in Tris-buffered saline containing 0.05% Tween 20 and 5% non-fat milk. The blot was incubated with the primary antibody at 4 ° C for 4-16 hours.
Tyrosine phosphorylated protein is detected with anti-phosphorylated antibody,
Expression of Myc-tagged HSH2 was detected by anti-myc antibody. pc
DNA3-myc (Mock) and pcDNA3-SHD (SHD) were used as controls. After the primary antibody reaction, a secondary antibody reaction was performed, followed by visualization using enhanced Luminol reagent (NEN).

FIG. 3 shows the results. SHD is the SH2 protein used as a control, and was phosphorylated by c-ABL, BCR-ABL, and v-Src. On the other hand, HSH2 is c-ABL, BCR-ABL, v-Sr
c, phosphorylated by JAK1, JAK2, JAK3. From the above, it was revealed that HSH2 is a substrate for the tyrosine kinase, suggesting that it may be involved in the signal transduction system via the tyrosine kinase.

Example 9 Binding of HSH2 to IL-3 Receptor β Chain Phosphorylated by IL-3 Stimulation and SHP2 Using the plasmid (pGEX SH2 / HSH2) prepared in Example 7, E. coli was made to produce the SH2 domain of HSH2 fused with, and purified with glutathione sepharose. Specifically, the procedure was performed as follows.

E. coli JM10 transformed with pGEX plasmid
9 was inoculated into 20 ml of LB medium containing 100 μg / ml of ampicillin, and cultured at 37 ° C. overnight. Culture the culture with 200 ml of LB Amp medium
Diluted at a ratio of 1:10 to 25, until the OD ₆₀₀ reaches 0.8.
Incubated at ℃. Thereafter, GST-fusion protein was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. Escherichia coli was recovered after incubation at 25 ° C. for 20 hours in the presence of IPTG, and resuspended in 20 ml of PBS containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF). After sonication of the E. coli suspension, the cells were incubated at 4 ° C and 30% in the presence of 1% Triton X-100.
Incubated for minutes. The suspension was centrifuged, the supernatant was incubated with glutathione Sepharose 4B (Pharmacia), and the GST-fusion protein was immobilized on Sepharose beads and purified. This was incubated with the IL-3 treated BaF3 cell extract to recover proteins that bind to the SH2 domain of HSH2. A lysate from BaF3 cells stimulated with IL-3 was prepared as follows.

Collecting exponentially growing BaF3 cells,
After washing twice with RPMI1640, the cells were resuspended in RPMI1640 containing 0.5% fetal bovine serum albumin at a concentration of 5 × 10 ⁵ cells / ml. 37
After incubating at 8 ° C for 8 hours, BaF3 cells were stimulated with IL-3 for 12.5 minutes, washed twice, and then NP- at a concentration of 2.5 x 10 ⁷ cells / ml.
It was dissolved in 40 lysis buffer. GST-fusion protein-Sepharose beads (about 2 μg of GST fusion protein) were incubated with cell lysate at 4 ° C. for 4 hours, and 0.1% Trit
The plate was washed twice with PBS containing on X-100 and twice with NP-40 lysis buffer. SDS-PAGE of the protein bound to Sepharose,
Anti-phosphorylated tyrosine antibody, anti-IL-3 receptor β-chain antibody, anti-SH
Western blot analysis was performed with the P2 antibody. Western blot analysis was performed in the same manner as in Example 8.

FIG. 4 shows the results. The SH2 domain of HSH2 is I
L-3 treatment was found to bind to the tyrosine-phosphorylated IL-3 receptor β-chain and SHP2. Therefore, HSH2 becomes IL
-3 suggests that it is involved in the hematopoietic signal transduction system.

[Example 10] Binding of HSH2 to tyrosine phosphorylated protein in K562 cell extract A cell lysate from K562 cells established from a chronic myeloid leukemia patient was prepared as follows. Collect logarithmically growing 1 × 10 ⁷ cells, wash twice with PBS, 2 × 4 ° C.
Incubate further for 0 min, and add 1m of NP-40 lysis buffer
dissolved in l. The cell lysate was transferred to a 1.5 ml tube and centrifuged.

The SH2 domain of HSH2 fused to GST was incubated with a K562 cell lysate to recover a protein binding to the SH2 domain of HSH2. SDS-PAGE of bound protein
Then, Western blot analysis was performed using an anti-phosphorylation antibody.

FIG. 5 shows the results. The SH2 domain of HSH2 was found to interact with multiple tyrosine phosphorylated proteins in K562 cells. From the size of the band, the phosphorylated proteins bound to HSH2 were BCR-ABL and C
It was expected that it was a molecule involved in information transmission such as BL and SHC.

Example 11 Binding of HSH2 to BCR-ABL by Yeast Two Hybrid Assay A yeast Two Hybrid assay was performed to examine whether HSH2 binds to BCR-ABL. The yeast TwoHybrid assay was performed as follows.

The L40 yeast strain was transformed with the pBTM plasmid (ABL WT;
BTM ABL WT (KpnI-BclI), ABL KD; pBTM ABL KD (KpnI-B
clI), p185bcr-abl WT; pBTM p185bcr-abl WT, p185bcr
-abl KD; pBTM p185bcr-abl KD)
Transfer the yeast strain to the pGAD plasmid (HSH2 full; pGAD-HSH2, SH
2 / HSH2; pGAD-SH2 / HSH2, ΔSH2 / HSH2; pGAD-ΔSH2 / HSH
2) Alternatively, pVP16 plasmid (CrkL; pVP16-CrkL, Grb
2; pVP16-Grb2). The construction of the plasmid is shown in Example 7 above. The transformed L40 and AMR70 were mixed and bound, and incubated on a YPD plate at 30 ° C. for 16 hours. Diploid yeast clones were selected and subjected to β-galactosidase assay.

FIG. 6 shows the results. Full length HSH2 and SH of HSH2
The two domains bound to wild-type BCR-ABL, but did not bind to HSH2 except for the SH2 domain. Also, no HSH2 bound to BCR-ABL without kinase activity. These results revealed that HSH2 directly binds to autophosphorylated BCR-ABL via its SH2 domain.

[0083]

Industrial Applicability According to the present invention, a novel human SH2 protein HSH2 involved in the tyrosine kinase-mediated signal transduction system, in particular, the differentiation, proliferation and activation of blood cells is provided. HSH2 binds to BCR-ABL, a gene product of chronic myeloid leukemia, and undergoes phosphorylation. BCR-ABL for chronic myeloid leukemia
It is thought that unregulated phosphorylation by gene products disrupts intracellular signal transduction and causes cancer. HS
Since H2 has a structure and function as a molecule involved in signal transduction, abnormalities in HSH2 function caused by BCR-ABL may be involved in disease development. Therefore, the HSH2 of the present invention
It is expected that a drug targeting, or a drug created based on the structure of HSH2, will act as an anticancer drug.

HSH2 is strongly expressed in blood cells and is phosphorylated by cytokine-activated JAK kinase. In addition, it binds to IL-3 receptor and SHP2 phosphorylated by IL-3 stimulation. This suggests that HSH2 may be involved in blood cell differentiation and proliferation. Therefore,
Drugs targeting HSH2 or drugs created based on the structure of HSH2 are expected to be drugs against hematopoiesis and blood diseases.

[Sequence List] SEQUENCE LISTING <110> Helix Research Institute <120> Novel human SH2 protein. <130> H1-107DP5 <140> <141> <150> JP 1999-248036 <151> 1999-07-29 <150> H1-107DP3 <151> 2000-05-02 <160> 14 <170> PatentIn Ver. 2.0 <210> 1 <211> 1916 <212> DNA <213> Homo sapiens <220> <221> CDS <222> ( 112) .. (1167) <400> 1 gtgcagacct tgaatcgaaa cccaggctcc tgcaggcact ggcacagcta cagcgagggc 60 ctcggccatc caagggtctc ccaggtgacc ttccctccac cccaggaagc t atg acag gc cg gc cg gc cg gc cg gc cg gc cg gc cg gc cg gcc gcc Lys Leu Pro Leu Pro Leu Pro Pro Arg Leu Asp Trp Phe 5 10 15 gtg cac acc cag atg ggc cag ctg gcc caa gac ggg gtc ccc gag tgg 213 Val His Thr Gln Met Gly Gln Leu Ala Gln Asp Gly Val Pro Glu Trp 20 25 30 ttc cat ggt gca atc tca aga gag gat gct gag aac ttg ctg gag tca 261 Phe His Gly Ala Ile Ser Arg Glu Asp Ala Glu Asn Leu Leu Glu Ser 35 40 45 50 cag cca ctg gga tcc ttt ctc atc agg gtc agt cac agc cat gtg ggc 309 Gln Pro Leu Gly Ser Phe Leu Ile Arg Val Ser His Ser His Val Gly 55 60 65 tac aca ctc tcc tac aaa gcc caa agc agc tgc tgc cat ttc atg gtg 357 Tyr Thr Leu Ser Tyr Lys Ala Gln Ser Ser Cys Cys His Phe Met Val 70 75 80 aag ctc ttg gat gat ggg act ttc atg atc ccc ggg gag aag gtg gcc 405 Lys Leu Leu Asp Asp Gly Thr Phe Met Ile Pro Gly Glu Lys Val Ala 85 90 95 cac acc tcg ctg gac gcc ctg gtc acc ttc cac cag cag aag cca att 453 His Thr Ser Leu Asp Ala Leu Val Thr Phe His Gln Gln Lys Pro Ile 100 105 110 gag ccg cgc agg gag ctg ctg aca cag ccc tgc agg cag aag gat ccc 501 Glu Pro Arg Arg Glu Leu Leu Thr Gln Pro Cys Arg Gln Lys Asp Pro 115 120 125 130 gca aac gtg gat tac gag gat ctc ttc ctc tac tcc aac gca gtg gcc 549 Ala Asn Val Asp Tyr Glu Asp Leu Phe Leu Tyr Ser Asn Ala Val Ala 135 140 145 gag gaa gct gcc tgc ccg ctg tct gag gag gcc tcc cca aag 597 Glu Glu Ala Ala Cys Pro Val Ser Ala Pro Glu Glu Ala Ser Pro Lys 150 155 160 cca gtc ctg tgt cac caa tca aag gaa agg aag ccg tca gca gag atg 645 Pro Val Leu Cys His Gln Ser Lys Glu Arg Lys Pro Ser Ala Glu Met 165 170 175 aac aga ata acc acc aag gaa gcc act tcc tcc tgc ccc cca aaa tcc 693 Asn Arg Ile Thr Thr Lys Glu Ala Thr Ser Ser Cys Pro Pro Lys Ser 180 185 190 cct ctt gga gag acc cgc cag aaa ctc tgg agg agc ctc aaa atg ctc 741 Pro Leu Gly Glu Glu Thr Arg Gln Lys Leu Trp Arg Ser Leu Lys Met Leu 195 200 205 210 ccc gag aga ggc cag agg gtc cgg cag cag cta aaa agc cacct Pro Glu Arg Gly Gln Arg Val Arg Gln Gln Leu Lys Ser His Leu Ala 215 220 225 act gtg aac ttg tcg tca ctc ttg gat gtc cgg aga tcc acg gtg atc 837 Thr Val Asn Leu Ser Ser Leu Leu Asp Val Arg Arg Ser Thr Val Ile 230 235 240 tca ggc cct ggg acc gga aaa ggc agc caa gat cac tca ggg gat ccc 885 Ser Gly Pro Gly Thr Gly Lys Gly Ser Gln Asp His Ser Gly Asp Pro 245 250 255 acc tcg ggg gac aga ggc tac acg gat ccc tgt gtg gcc aca tct ctc 933 Thr Ser Gly Asp Arg Gly Tyr Thr Asp Pro Cys Val Ala Thr Ser Leu 260 265 270 aaa agc ccc tca cag ccc cag gca cca aaa gac aga aag gtc ccc acc 981 Lys Ser Pro Ser Gln ProGln Ala Pro Lys Asp Arg Lys Val Pro Thr 275 280 285 290 agg aag gcc gag agg tcg gtc agc tgc att gag gtg acc cca ggg gac 1029 Arg Lys Ala Glu Arg Ser Val Ser Cys Ile Glu Val Thr Pro Gly Asp 295 300 305 agg agt tgg cac caa atg gta gtg aga gcc cta tcc tcc cag gag tcc 1077 Arg Ser Trp His Gln Met Val Val Arg Ala Leu Ser Ser Gln Glu Ser 310 315 320 aag cca gag cac cag ggc ttg gca gag cct gag aac gac cag ctc ccg 1125 Lys Pro Glu His Gln Gly Leu Ala Glu Pro Glu Asn Asp Gln Leu Pro 325 330 335 gag gag tac caa caa ccg cca ccc ttt gcc cct ggg tac tgc 1167 Glu Glu Tyr Gln Gln Pro Pro Pro Phe Ala Pro Gly Tyr Cys 340 345 350 tagagaacag gtccaccctg gctctgggac tcgctgccag gggctgccac actcctgaat 1227 gccttaacat ttcttccatg gccccacacc atggcatccg ggggtcttcg ggaacccggg 1287 aaatggaata aagatgtttt tggggtctgt tcctgcactc acccatgggg tgagctggtt 1347 attttagcaa caatcatcag agtgacgctg atggtttggg gcaccagcta tacatcagcc 1407 ccagtgccag accttctatt cattatttta cgcctcagag caaggccctc agggagggtc 1467 atcctccatg ttttgaagaa gag actgagg ttcagagagg ataagaggcg tgaccaaggc 1527 cacagagcta tgggtgtcag caccaggatt tgaagccagg tgaatccgag cccttttccc 1587 atatcatctg tttgttctgt tgtctaaaag cacactgcaa gccgggctca gtggctcatg 1647 cctgtagtcc cagcactctg tggggccgag gcaggcagat cgcttgaggt caggagtttg 1707 agaccagcct ggccaacatg gtgaaacccc gtctatacta aaaaattcaa aaattacccg 1767 gacgtggtgg cgcatgcctg taatcccagc tacttgggag cctgaggcgg gagaattgct 1827 tgaacccggg aggcagaggt tgcagtgagc cgagatcgca tcactgcagt ccagcctgga 1887 tgacagagtg agactccatc tcaaaaaat 1916 <210> 2 <211> 352 <212> PRT <213> Homo sapiens <400> 2 Met Thr Glu Ala Gly Lys Leu Pro Leu Pro Leu Pro Pro Arg Leu Asp 1 5 10 15 Trp Phe Val His Thr Gln Met Gly Gln Leu Ala Gln Asp Gly Val Pro 20 25 30 Glu Trp Phe His Gly Ala Ile Ser Arg Glu Asp Ala Glu Asn Leu Leu 35 40 45 Glu Ser Gln Pro Leu Gly Ser Phe Leu Ile Arg Val Ser His Ser His 50 55 60 Val Gly Tyr Thr Leu Ser Tyr Lys Ala Gln Ser Ser Cys Cys His Phe 65 70 75 80 Met Val Lys Leu Leu Asp Asp Gly Thr Phe Met Ile Pro Gly Glu Lys 85 90 95 Val Ala His Thr Ser Leu Asp Ala Leu Val Thr Phe His Gln Gln Lys 100 105 110 Pro Ile Glu Pro Arg Arg Glu Leu Leu Thr Gln Pro Cys Arg Gln Lys 115 120 125 Asp Pro Ala Asn Val Asp Tyr Glu Asp Leu Phe Leu Tyr Ser Asn Ala 130 135 140 Val Ala Glu Glu Ala Ala Cys Pro Val Ser Ala Pro Glu Glu Ala Ser 145 150 155 160 Pro Lys Pro Val Leu Cys His Gln Ser Lys Glu Arg Lys Pro Ser Ala 165 170 175 Glu Met Asn Arg Ile Thr Thr Lys Glu Ala Thr Ser Ser Cys Pro Pro 180 185 190 Lys Ser Pro Leu Gly Glu Thr Arg Gln Lys Leu Trp Arg Ser Leu Lys 195 200 205 Met Leu Pro Glu Arg Gly Gln Arg Val Arg Gln Gln Leu Lys Ser His 210 215 220 Leu Ala Thr Val Asn Leu Ser Ser Leu Leu Asp Val Arg Arg Ser Thr 225 230 235 240 Val Ile Ser Gly Pro Gly Thr Gly Lys Gly Ser Gln Asp His Ser Gly 245 250 255 Asp Pro Thr Ser Gly Asp Arg Gly Tyr Thr Asp Pro Cys Val Ala Thr 260 265 270 270 Ser Leu Lys Ser Pro Ser Gln Pro Gln Ala Pro Lys Asp Arg Lys Val 275 280 285 Pro Thr Arg Lys Ala Glu Arg Ser Val Ser Cys Ile Glu Val Thr Pro 290 295 Three 00 Gly Asp Arg Ser Trp His Gln Met Val Val Arg Ala Leu Ser Ser Gln 305 310 315 320 Glu Ser Lys Pro Glu His Gln Gly Leu Ala Glu Pro Glu Asn Asp Gln 325 330 335 Leu Pro Glu Glu Tyr Gln Gln Pro Pro Pro Phe Ala Pro Gly Tyr Cys 340 345 350 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Sequence <400> 3 agcacgagcg gccgggccac gg 22 <210 > 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 4 gcggctgaag acggcctatg tggccttttt tttttttttt tt 42 <210> 5 <211> 21 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 5 agcatcgagt cggccttgtt g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 6 gcggctgaag acggcctatg t 21 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 7 gagagaattc atgacagagg ccgggaagct g 31 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 8 gagactcgag ctagcagtac ccaggggcaa a 31 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400 > 9 gagagaattc atgacagagg ccgggaagct g 31 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 10 gagactcgag ctactcgtaa tccacgtttg cggg 34 <210 > 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Sequence <400> 11 tcgactggaa ttcggatccg tcgacctcga ga 32 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artifi cially Synthesized Sequence <400> 12 aatttctcga ggtcgacgga tccgaattcc ag 32 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 13 gagagaattc cccgcaaacg tggattacga 30 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially Synthesized Primer Sequence <400> 14 gagactcgag ctactcgtaa tccacgtttg cggg 34

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of the HSH2 protein of the present invention and the nucleotide sequence of the gene encoding it. The SH2 domain is underlined, the proline-rich region is indicated by a box, and tyrosine residues are indicated by circles.

FIG. 2 shows the results of Northern blot analysis. The 2.2 kb band in peripheral blood leukocytes was
A.

FIG. 3 shows the phosphorylation of HSH2 by tyrosine kinase. SHD stands for SH2 protein.

FIG. 4 is a diagram showing the binding of IL-3 receptor β-chain and SHP2 phosphorylated by IL-3 stimulation to HSH2 protein. "St
"arved" represents BaF3 cells not stimulated with IL-3.

FIG. 5 is a diagram showing the binding of tyrosine phosphorylated protein in the extract of K562 cells to HSH2 protein.

FIG. 6 shows the binding of HSH2 protein to BCR-ABL by the yeast Two Hybrid assay. When the protein shown in the vertical direction and the protein shown in the horizontal direction are bound, a band is visible at the position where the lane of each protein crosses.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C 5/10 21/08 C12P 21/02 G01N 33/15 Z 21/08 33/50 Z G01N 33/15 33/53 D 33/50 C12R 1:91) 33/53 (C12P 21/02 C // (C12N 5/10 C12R 1:91) C12R 1:91) (C12P 21 / 08 (C12P 21/02 C12R 1:91) C12R 1:91) C12N 15/00 ZNAA (C12P 21/08 5/00 A C12R 1:91) C12R 1:91) (72) Inventor Takao Isogai Inashiki, Ibaraki Prefecture 511-12 Omuro, Ami-gun, Gunma (72) Inventor Tetsuo Nishikawa 27-3-403, Hikawacho, Itabashi-ku, Tokyo (72) Inventor Koji Hayashi 1-9-446, Ariakidai Nishi, Ichihara City, Chiba Prefecture (72) Invention Person Kaoru Saito 2-2-1-1201 Kisarazu, Kisarazu-shi, Chiba (72) Invention Junichi Yamamoto 3-28-3-A 101, Kiyomidai-Higashi, Kisarazu-shi, Chiba 101 (72) Inventor Shizuko Ishii 4508-19-202, Yana, Kisarazu-shi, Chiba (72) Inventor Tomoyasu Sugiyama 2-6, Kiyomidai, Kisarazu-shi, Chiba 23-102 (72) Inventor Ai Wakamatsu 1473-4-202 Takayanagi, Kisarazu-shi, Chiba (72) Inventor Keiichi Nagai 3-44-14-9-204 (44) Inventor Tetsuji Otsuki Chiba Chiba 3-1-10-B102 Asahi, Kisarazu-shi, Pref.F-term (reference) AA90X AA93Y AB01 BA01 CA24 CA25 CA44 CA46 4H045 AA10 AA11 BA10 CA40 DA01 DA76 EA20 EA50 FA74

Claims (13)

[Claims] 1. The DNA according to any one of the following (a) to (d). (A) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. (B) DNA containing the coding region of the nucleotide sequence described in SEQ ID NO: 1. (C) a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and the amino acid sequence of SEQ ID NO: 2; DNA that encodes an equivalent protein. (D) a DNA which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2. 2. A DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO: 2. 3. A protein or peptide encoded by the DNA according to claim 1 or 2. 4. A vector into which the DNA according to claim 1 or 2 has been inserted. 5. A transformed cell carrying the DNA according to claim 1 or 2 or the vector according to claim 4. 6. A step of culturing the transformed cell according to claim 5, and recovering the expressed protein or peptide from the transformed cell or a culture supernatant thereof.
3. The method for producing a protein or peptide according to item 1. 7. An antibody that binds to the protein according to claim 3. 8. It consists of the nucleotide sequence of SEQ ID NO: 1.
A polynucleotide comprising at least 15 nucleotides complementary to DNA or its complementary strand. 9. A method for screening a compound that binds to a protein according to claim 3, wherein (a) contacting a test sample with the protein or a partial peptide thereof;
(B) detecting a binding activity between the protein or its partial peptide and a test sample; and (c) selecting a compound having an activity of binding to the protein or its partial peptide. 10. A method for screening a compound having an activity of regulating the phosphorylation of the protein according to claim 3 by a tyrosine kinase, wherein (a) the protein or a partial peptide thereof, a tyrosine kinase, and a test sample (B) detecting phosphorylation of the protein or its partial peptide, (c) detecting the degree of phosphorylation detected in step (b) in the absence of the test sample Selecting a compound that decreases or increases as compared to 11. The tyrosine kinase is c-ABL, BCR-AB
The method according to claim 10, wherein the method is selected from the group consisting of L, v-Src, JAK1, JAK2, and JAK3. 12. A method for screening a compound having an activity of regulating the binding between the protein according to claim 3 and a tyrosine phosphorylated protein, comprising: (a) the protein or a partial peptide thereof, a tyrosine phosphorylated protein; And contacting the test sample, (b) detecting the binding of the protein or its partial peptide to the tyrosine phosphorylated protein, and (c) determining the binding activity detected in the step (b). Selecting a compound that decreases or increases as compared to when detected in the absence. 13. The method of claim 12, wherein the tyrosine phosphorylated protein is selected from the group consisting of an IL-3 receptor β chain, SHP2, BCR-ABL, CBL, and SHC.